Proliferation and differentiation of hematopoietic cells are regulated by hormone-like growth and differentiation factors designated as colony-stimulating factors (CSF) (Metcalf, D. Nature 339, 27-30 (1989)). CSF can be classified into several factors according to the stage of the hematopoietic cells to be stimulated and the surrounding conditions as follows: granulocyte colony-stimulation factor (G-CSF), granulocyte-macrophage colony-stimulation factor (GM-CSF), macrophage colony-stimulation factor (M-CSF), and interleukin 3 (IL-3). G-CSF participates greatly in the differentiation and growth of neutrophilic granulocytes and plays an important role in the regulation or blood levels of neutrophils and the activation of mature neutrophils (Nagata, S., "Handbook of Experimental Pharmacology", volume "Peptide Growth Factors and Their Receptors", eds. Sporn, M. B. and Roberts, A. B., Spring-Verlag, Heidelberg, Vol.95/1, pp.699-722 (1990); Nicola, N. A. et al., Annu.Rev.Biochem. 58, pp.45-77 (1989)). Thus, G-CSF stimulates the growth and differentiation of neutrophilic granulocytes through the interaction between cell-surface receptors on precursors of neutrophilic granulocytes to give mainly the neutrophilic granulocytes (Nicola, N. A. & Metcalf, D., Proc. Natl. Acad., Sci. USA, 81, 3765-3769 (1984)).
G-CSF has various biological activities in addition to those mentioned above. For example, G-CSF prepared by recombinant DNA technology has proven to be a potent regulator of neutrophils in vivo using animal model systems (Tsuchiya et al., EMBO J. 6 611-616 (1987); and Nicola et al., Annu. Rev. Biochem. 58, 45-77 (1989)). Recent clinical trials in patients suffering from a variety of hemopoietic disorders have shown that the administration of G-CSF is beneficial in chemotherapy and bone marrow transplantation therapy (Morstyn et al., Trends Pharmacol. Sci. 10, 154-159 (1989)). It is also reported that G-CSF stimulates the growth of tumor cells such as myeloid leukemia cells.
Despite the biological and clinical importance of G-CSF, little is known about the mechanism through which G-CSF exerts its effects. Therefore, it has been needed to elucidate such mechanism to establish more effective treatment and diagnosis for G-CSF-related disorders. For this purpose, the biochemical characterization of specific cell-surface receptors for G-CSF and the evaluation of interaction between G-CSF and the receptor must be performed.
Several reports suggested that the target cells of G-CSF is restricted to progenitor and mature neutrophils and various myeloid leukemia cells (Nicola and Metcalf, Proc. Natl. Acad. Sci. USA, 81, 3765-3769 (1984); Begley et al., Leukemia, 1, 1-8 (1987); and Park et al., Blood 74, 56-65 (1989)). Human G-CSF is a 174 amino acid polypeptide while murine G-CSF consists of 178 amino acids. Human and mouse G-CSFs are highly homologous (72.6%) at the amino acid sequence level, in agreement with the lack of species-specificity between them (Nicola et al, Nature 314, 626-628 (1985)). What makes the research in G-CSF more interesting is that G-CSF receptor has also recently been found in non hemopoietic cells such as human endothelial cells (Bussolino et al., Nature 337, 471-473 (1989)) and placenta (Uzumaki et al., Proc. Natl. Acad. Sci. USA, 86, 9323-9326 (1989)).
As can be seen from the above, the elucidation of the interaction between G-CSF and its receptor should greatly contribute to the development of the treatment or prophylaxis of various diseases including hematopoietic disorders using G-CSF, whereby providing more effective and proper treatments on such diseases. Thus, such elucidation is important not only academically but also clinically. On the other hand, the receptor itself can be useful. For instance, a soluble form of the G-CSF receptor may be useful clinically to inhibit the proliferation of some G-CSF-dependent human myeloid leukemia cells (Santoli et al., J.Immunol. 139, 3348-3354 (1987)). The investigation into the expression of G-CSF receptor in tumor cells such as myeloid leukemia may be beneficial to establish an effective clinical application of G-CSF. Accordingly, owing to the various academic and practical usefulness, a stable supply of a G-CSF receptor-encoding gene and the G-CSF receptor has been demanded.
Recently, the technology of genetic engineering has been used for the production of various physiologically active substances. The production by the genetic engineering is generally carried out by cloning DNA encoding desired polypeptide, inserting said DNA into a suitable expression vector, transforming an appropriate host cell such as microorganism or animal cell by the expression vector, and making the transformant express the desired polypeptide.
To apply the genetic engineering technique to the production of G-CSF receptor, cloning of DNA encoding G-CSF receptor is firstly required. However, cloning cDNA encoding G-CSF receptor was hampered by the low number of receptors present on the cell surface (at most hundreds to 2,000 receptor per cell).